Chalcone-Derived Lactones: Synthesis, Whole-Cell Biotransformation, and Evaluation of Their Antibacterial and Antifungal Activity

Four compounds with lactone moiety were synthesized from chalcone 1 in three- or four-step synthesis. γ-Bromo-δ-lactone 5 was the only product of bromolactonization of acid 4 whereas bromolactonization of ester 3, apart from lactone 5 also afforded its isomer 6 and two diastereoisomeric δ-hydroxy-γ-lactones 7 and 8. Lactone 8 was also obtained in 88% yield as a product of simultaneous dehalogenation and translactonization of γ-bromo-δ-lactone 5 by Penicillum frequentans AM 359. Chalcone-derived lactones 5–8 were subjected to the tests on antimicrobial activity and the results compared with activity of starting chalcone 1. Obtained lactones 5–8 in most cases limited the growth of tested bacterial and fungal strains. The highest activity was found for δ-hydroxy-γ-lactone 8 which completely inhibited the growth of Staphylococcus aureus, Fusarium graminearum, Aspergillus niger, and Alternaria sp. The introduction of lactone moiety into chalcone scaffold significantly improved antimicrobial activity of the compound: γ-bromo-δ-lactone 6 and δ-hydroxy-γ-lactone 8 were significantly stronger growth inhibitors of S. aureus and F. graminearum. In the case of the latter, a clear positive effect of the lactone function on the antifungal activity was also observed for γ-bromo-δ-lactone 5.


Introduction
Chalcones (1,3-diarylprop-2-en-1-ones) are secondary metabolites of plants which belong to the class of flavonoids. They are widely distributed in vegetables, fruits, and teas [1]. Some plants rich in chalcones from the Glycyrrhiza, Piper, or Angelica genus have been used for many years as therapeutic agents in Balkan countries [2]. Indeed, for natural and synthetic chalcones, a number of studies have shown a wide spectrum of biological activity of such as antioxidant, anticancer, anti-inflammatory, antidiabetic, antiviral, or antiparasitic [2][3][4][5]. Several chalcone derivatives are known to be the commercial drugs, i.a., choleretic drug metochalcone or antiulcer and mucoprotective drug sofalcone [6].
Our research group was also involved in the studies on the lactones with antimicro bial activity, derived from simple aromatic aldehydes [17][18][19] or β-cyclocitral [20]. In on of our last papers, we presented the results of the studies on the antimicrobial propertie of ε-lactones obtained from flavanones by Baeyer-Villiger oxidation. It has been con firmed that introduction of lactone moiety increased the antimicrobial activity of startin flavanones [21]. In this work, we would like to investigate if the lactones obtained from chalcone 1 by synthetic and biotechnological methods exhibit higher antimicrobial prop erties than starting substrate.

Synthesis
Direct substrates for halolactonizations were acid 4 or ethyl ester 3. Both substrate were obtained from chalcone 1 in two or three-step procedure presented on Scheme 1. I the first step, chalcone 1 was reduced with sodium borohydride in methanol-water solu tion (10:1) at 0 °C to afford racemic alcohol 2 in 97% yield. Alcohol 2 was then subjected t the Claisen rearrangement with triethyl orthoacetate in the presence of catalytic amoun of propionic acid to obtain ethyl ester 3 in 70% yield. Ester 3 was subsequently hydrolyze with NaOH in EtOH to afford acid 4 in 81% yield. All spectroscopic data of alcohol 2, este 3, and acid 4 were in accordance with those reported in literature [22,23]. Bromolactonization of acid 4 with N-bromosuccinimide (NBS) in tetrahydrofuran (THF) afforded γ-bromo-δ-lactone 5 as the only product in 37% yield (Scheme 2). Its stru ture was confirmed by spectroscopic data. On IR spectrum, the absorption bands at 172 and 1249 cm −1 of corresponding C=O and C-O bonds confirmed the presence of δ-lacton ring in the molecule. On 1 H NMR, two diastereotopic protons of CH2-3 group were repre sented by two doublets of doublets at 2.90 ppm (J = 17.8 and 10.1 Hz) and 3.19 ppm (J 17.8 and 6.7 Hz). The values of the smaller coupling constants in these multiplets let u assign the multiplet located at higher field to pseudoaxial proton and the multiplet locate at lower field to the pseudoequatorial one. Bromolactonization of acid 4 with N-bromosuccinimide (NBS) in tetrahydrofurane (THF) afforded γ-bromo-δ-lactone 5 as the only product in 37% yield (Scheme 2). Its structure was confirmed by spectroscopic data. On IR spectrum, the absorption bands at 1729 and 1249 cm −1 of corresponding C=O and C-O bonds confirmed the presence of δ-lactone ring in the molecule. On 1 H NMR, two diastereotopic protons of CH 2 -3 group were represented by two doublets of doublets at 2.90 ppm (J = 17.8 and 10.1 Hz) and 3.19 ppm (J = 17.8 and 6.7 Hz). The values of the smaller coupling constants in these multiplets let us assign the multiplet located at higher field to pseudoaxial proton and the multiplet located at lower field to the pseudoequatorial one. Proton H-4 gave triplet (J = 10.1 Hz) of doublets (J = 6.7 Hz) at 3.63 ppm, proton H-5 triplet (J = 10.1 Hz) at 4.30 ppm, and the signal of proton H-6 was recognized as doublet at 5.47 ppm (J = 10.1 Hz). The coupling constant value (10.1 Hz) between proton H-5 and H-4 as well as H-5 and H-6 indicated the pseudoaxial orientations of these protons and thus the pseudoequatorial orientations of bromine and two phenyl rings. These data were fully confirmed by X-ray analysis. Crystal structure of compound 5, shown in Figure 1, revealed trans orientation of bromine at C-5 in relation to phenyl substituents at C-4 and C-6 as well as the half-chair conformation of δ-lactone ring, assumed on the basis of spectroscopic data. The values of torsion angles between corresponding bonds were compatible with the coupling constants found in the 1 H NMR spectrum. The spectroscopic data of lactone 5 were consistent with those obtained for its enantiomer 4R,5R,6S, an intermediate in the synthesis of (−)clausenamide, exhibiting significant neuroprotective effect against β-amyloid in cellular models [23].
Molecules 2023, 28, x FOR PEER REVIEW 3 of 19 value (10.1 Hz) between proton H-5 and H-4 as well as H-5 and H-6 indicated the pseudoaxial orientations of these protons and thus the pseudoequatorial orientations of bromine and two phenyl rings. These data were fully confirmed by X-ray analysis. Crystal structure of compound 5, shown in Figure 1, revealed trans orientation of bromine at C-5 in relation to phenyl substituents at C-4 and C-6 as well as the half-chair conformation of δ-lactone ring, assumed on the basis of spectroscopic data. The values of torsion angles between corresponding bonds were compatible with the coupling constants found in the 1 H NMR spectrum. The spectroscopic data of lactone 5 were consistent with those obtained for its enantiomer 4R,5R,6S, an intermediate in the synthesis of (˗)-clausenamide, exhibiting significant neuroprotective effect against β-amyloid in cellular models [23].  Searching for new methods to improve the yield of lactone 5, bromolactonization of ester 3 using NBS in THF/H2O solution was carried out (Scheme 2) according to the procedure described by Obara et al. [24]. As a result, we obtained bromolactone 5 as a major product with a significantly higher isolated yield (79%) compared to the yield observed for the bromolactonization of acid 3. Simultaneously, three new lactones, not described previously, were formed as minor products: bromolactone 6 (4% isolated yield) and two δ-hydroxy-γ-lactones 7 and 8, obtained in 3 and 6% isolated yields, respectively. Searching for new methods to improve the yield of lactone 5, bromolactonization of ester 3 using NBS in THF/H 2 O solution was carried out (Scheme 2) according to the procedure described by Obara et al. [24]. As a result, we obtained bromolactone 5 as a major product with a significantly higher isolated yield (79%) compared to the yield observed for the bromolactonization of acid 3. Simultaneously, three new lactones, not described previously, were formed as minor products: bromolactone 6 (4% isolated yield) and two δ-hydroxy-γ-lactones 7 and 8, obtained in 3 and 6% isolated yields, respectively. Spectral data obtained for lactone 6 together with X-ray analysis allowed us to establish the structure of this product. The presence of δ-lactone ring was confirmed by absorption bands at 1730 and 1205 cm −1 on the IR spectrum. The orientations of substituents at the lactone ring were clearly indicated by the crystal structure ( Figure 2).

Bond
Torsion angle (°)  Spectral data obtained for lactone 6 together with X-ray analysis allowed us to establish the structure of this product. The presence of δ-lactone ring was confirmed by absorption bands at 1730 and 1205 cm −1 on the IR spectrum. The orientations of substituents at the lactone ring were clearly indicated by the crystal structure ( Figure 2). Spectral data obtained for lactone 6 together with X-ray analysis allowed us to establish the structure of this product. The presence of δ-lactone ring was confirmed by absorption bands at 1730 and 1205 cm −1 on the IR spectrum. The orientations of substituents at the lactone ring were clearly indicated by the crystal structure ( Figure 2).

Bond
Torsion angle (°)   One can see pseudoaxial orientations of bromine atom at C-5 and phenyl ring at C-6 as well as pseudoequatorial orientation of phenyl ring at C-4, which contrary to the lactone 5 was cis oriented to the bromine atom. The torsion angles between proton H-5 and protons H-4 and H-6 ( Figure 2) were in accordance with the corresponding coupling constants found in 1 H NMR spectrum. Similar to lactone 5, the signals of H-5 and H-6 were also triplet (4.71 ppm) and doublet (5.97 ppm), respectively, but with small coupling constant (J = 2.5 Hz) resulting from the pseudoequatorial orientations of these protons.
Based on the previous investigations on the bromolactonization of γ,δ-unsaturated acids [24][25][26], we also expected the formation of two diastereoisomeric δ-bromo-γ-lactones. Indeed, two other products were isolated from the products mixture but surprisingly, their structural X-ray analysis did not confirm their structures as predicted cis and trans isomers of δ-bromo-γ-lactone. The crystal structure of compound 7 ( Figure 3) undoubtedly showed that the isolated compound was trans δ-hydroxy-γ-lactone. One can see pseudoaxial orientations of bromine atom at C-5 and phenyl ring at C-6 as well as pseudoequatorial orientation of phenyl ring at C-4, which contrary to the lactone 5 was cis oriented to the bromine atom. The torsion angles between proton H-5 and protons H-4 and H-6 ( Figure 2) were in accordance with the corresponding coupling constants found in 1 H NMR spectrum. Similar to lactone 5, the signals of H-5 and H-6 were also triplet (4.71 ppm) and doublet (5.97 ppm), respectively, but with small coupling constant (J = 2.5 Hz) resulting from the pseudoequatorial orientations of these protons.
Based on the previous investigations on the bromolactonization of γ,δ-unsaturated acids [24][25][26], we also expected the formation of two diastereoisomeric δ-bromo-γ-lactones. Indeed, two other products were isolated from the products mixture but surprisingly, their structural X-ray analysis did not confirm their structures as predicted cis and trans isomers of δ-bromo-γ-lactone. The crystal structure of compound 7 (Figure 3) undoubtedly showed that the isolated compound was trans δ-hydroxy-γ-lactone.

Bond
Torsion angle (º) H3c-C3-C4-H4 9.62 H3t-C3-C4-H4 110.60 H4-C4-C5-H5 107.09 H5-C5-C6-H6 71.84 c-cis in a relation to H-4, t-trans in a relation to H-4. On the IR spectrum of lactone 8, apart from the presence of γ-lactone ring (absorption bands at 1778 and 1199 cm −1 ), a strong band at 3435 cm −1 from OH group was also detected. In determining the detailed structure of this compound, it was very helpful to compare its 1 H NMR data with those of the hydroxylactone obtained from benzaldehyde [27], which is a structural analog containing a methyl group at C-6 instead of a phenyl ring (Table 1). Based on the far-reaching similarities in the chemical shifts of the selected protons and the coupling constants found in the individual multiplets, particularly between H-4 and H-5 (J = 6.3 Hz), it was proven that product 8 is also a trans isomer of δ-hydroxy-γ-lactone. On the IR spectrum of lactone 8, apart from the presence of γ-lactone ring (absorption bands at 1778 and 1199 cm −1 ), a strong band at 3435 cm −1 from OH group was also detected. In determining the detailed structure of this compound, it was very helpful to compare its 1 H NMR data with those of the hydroxylactone obtained from benzaldehyde [27], which is a structural analog containing a methyl group at C-6 instead of a phenyl ring (Table 1). Based on the far-reaching similarities in the chemical shifts of the selected protons and the coupling constants found in the individual multiplets, particularly between H-4 and H-5 (J = 6.3 Hz), it was proven that product 8 is also a trans isomer of δ-hydroxy-γ-lactone. Table 1. Comparison of selected signals on 1 H NMR spectra (chemical shifts, coupling constants) for trans δ-hydroxy-γ-lactone 8 and its analog derived from benzaldehyde [27].
trans δ-hydroxy-γ-lactone derived from benzaldehyde Molecules 2023, 28, x FOR PEER REVIEW 6 of 19 Table 1. Comparison of selected signals on 1 H NMR spectra (chemical shifts, coupling constants) for trans δ-hydroxy-γ-lactone 8 and its analog derived from benzaldehyde [27]. In both isolated hydroxylactones 7 and 8, a significant difference was observed for chemical shift of proton H-6; the doublet from this proton was located at 4.76 ppm on the spectrum of compound 8 but shifted downfield to 5.13 ppm on the spectrum of lactone 7. This can be explained by the shorter distance between proton H-6 and the alkoxy oxygen of lactone ring ( Figure 3) which exerts more deshielding effect on H-6 in the molecule of lactone 7. For lactone 8, this effect is weaker which must be caused by a longer distance between H-6 and O1. The obtained data showed that both trans δ-hydroxy-γ-lactones 7 and 8 are diastereoisomers on C-6, differing in the position of the OH group in the relation to the C-O bond of the lactone ring. Formation of two products during bromolactonization of ester 3 can be explained by low stability of C-Br bond in a benzylic position of initially formed trans δ-bromo-γ-lactone and nucleophilic substitution occurring according to the SN1 mechanism (Scheme 3). In the reaction conditions after dissociation of bromine, the formed carbocation reacts easily with water to form two diastereoisomeric hydroxylactones 7 and 8. As the water molecule can approach both sides of planar carbocation, in lactone 7, the OH group and C-O bond of the lactone ring reside on opposite faces of the plane defined by the C5-C6 bond (location anti in which torsion angle O1-C5-C6-O2 is in the range of 150-180°), whereas in the lactone 8, they are situated on the same faces of this plane (location syn in which torsion angle O1-C5-C6-O2 is in the range of 0-30°) ( Figure  4). These structural features cause the difference in chemical shift of H-6 observed on 1 H NMR spectra as described above.
trans δ-hydroxy-γ-lactone 8 Molecules 2023, 28, x FOR PEER REVIEW 6 of 19 Table 1. Comparison of selected signals on 1 H NMR spectra (chemical shifts, coupling constants) for trans δ-hydroxy-γ-lactone 8 and its analog derived from benzaldehyde [27]. In both isolated hydroxylactones 7 and 8, a significant difference was observed for chemical shift of proton H-6; the doublet from this proton was located at 4.76 ppm on the spectrum of compound 8 but shifted downfield to 5.13 ppm on the spectrum of lactone 7. This can be explained by the shorter distance between proton H-6 and the alkoxy oxygen of lactone ring ( Figure 3) which exerts more deshielding effect on H-6 in the molecule of lactone 7. For lactone 8, this effect is weaker which must be caused by a longer distance between H-6 and O1. The obtained data showed that both trans δ-hydroxy-γ-lactones 7 and 8 are diastereoisomers on C-6, differing in the position of the OH group in the relation to the C-O bond of the lactone ring. Formation of two products during bromolactonization of ester 3 can be explained by low stability of C-Br bond in a benzylic position of initially formed trans δ-bromo-γ-lactone and nucleophilic substitution occurring according to the SN1 mechanism (Scheme 3). In the reaction conditions after dissociation of bromine, the formed carbocation reacts easily with water to form two diastereoisomeric hydroxylactones 7 and 8. As the water molecule can approach both sides of planar carbocation, in lactone 7, the OH group and C-O bond of the lactone ring reside on opposite faces of the plane defined by the C5-C6 bond (location anti in which torsion angle O1-C5-C6-O2 is in the range of 150-180°), whereas in the lactone 8, they are situated on the same faces of this plane (location syn in which torsion angle O1-C5-C6-O2 is in the range of 0-30°) ( Figure  4). These structural features cause the difference in chemical shift of H-6 observed on 1 H NMR spectra as described above. In both isolated hydroxylactones 7 and 8, a significant difference was observed for chemical shift of proton H-6; the doublet from this proton was located at 4.76 ppm on the spectrum of compound 8 but shifted downfield to 5.13 ppm on the spectrum of lactone 7. This can be explained by the shorter distance between proton H-6 and the alkoxy oxygen of lactone ring ( Figure 3) which exerts more deshielding effect on H-6 in the molecule of lactone 7. For lactone 8, this effect is weaker which must be caused by a longer distance between H-6 and O1. The obtained data showed that both trans δ-hydroxy-γ-lactones 7 and 8 are diastereoisomers on C-6, differing in the position of the OH group in the relation to the C-O bond of the lactone ring. Formation of two products during bromolactonization of ester 3 can be explained by low stability of C-Br bond in a benzylic position of initially formed trans δ-bromo-γ-lactone and nucleophilic substitution occurring according to the S N 1 mechanism (Scheme 3). In the reaction conditions after dissociation of bromine, the formed carbocation reacts easily with water to form two diastereoisomeric hydroxylactones 7 and 8. As the water molecule can approach both sides of planar carbocation, in lactone 7, the OH group and C-O bond of the lactone ring reside on opposite faces of the plane defined by the C5-C6 bond (location anti in which torsion angle O1-C5-C6-O2 is in the range of 150-180 • ), whereas in the lactone 8, they are situated on the same faces of this plane (location syn in which torsion angle O1-C5-C6-O2 is in the range of 0-30 • ) ( Figure 4). These structural features cause the difference in chemical shift of H-6 observed on 1 H NMR spectra as described above. Scheme 3. Proposed mechanism of formation of two diastereoisomeric trans δ-hydroxy-γ-lactones 7 and 8 from δ-bromo-γ-lactone.

Biotransformations of Bromolactone 5
The biotransformation of halolactones has been investigated in our research group for many years since it is an alternative to produce new lactone derivatives, usually difficult to obtain by chemical synthesis. γ-Bromo-δ-lactone 5 was also subjected to biotransformation with whole cells of filamentous fungi and yeasts. The preliminary screening showed that 12 strains transformed the substrate to only one product and let us estimate the effectiveness of used strains by comparing the substrate conversions and times of transformation required to achieve the highest conversion (Table 2).

Biotransformations of Bromolactone 5
The biotransformation of halolactones has been investigated in our research group for many years since it is an alternative to produce new lactone derivatives, usually difficult to obtain by chemical synthesis. γ-Bromo-δ-lactone 5 was also subjected to biotransformation with whole cells of filamentous fungi and yeasts. The preliminary screening showed that 12 strains transformed the substrate to only one product and let us estimate the effectiveness of used strains by comparing the substrate conversions and times of transformation required to achieve the highest conversion (Table 2).

Biotransformations of Bromolactone 5
The biotransformation of halolactones has been investigated in our research group for many years since it is an alternative to produce new lactone derivatives, usually difficult to obtain by chemical synthesis. γ-Bromo-δ-lactone 5 was also subjected to biotransformation with whole cells of filamentous fungi and yeasts. The preliminary screening showed that 12 strains transformed the substrate to only one product and let us estimate the effectiveness of used strains by comparing the substrate conversions and times of transformation required to achieve the highest conversion (Table 2). Among tested strains, the lowest conversion (56%) was observed for A. niger MB after 14 days of process (entry 1). A. niger 13/33 converted the substrate in 74% after 14 days (entry 2) whereas the conversion observed in A. niger 13/5 and A. niger KB cultures was 88 and 85% after 3 or 14 days, respectively (entries 3, 4). Complete transformation of lactone 5 into the product was achieved for eight strains: in the case of A. niger SBJ and A. niger SBP after 14 days (entries 5, 6), in the case of yeasts R. marina AM77 and fungal strains P. chrysogenum AM 112, P. chermesinum AM 113, A. glauca AM 254 after 10 days (entries 7-10). The most effective biocatalysts were P. frequentans AM 359, and A. niger CH 11/21 which transformed substrate completely after 7 days of incubation (entries 11,12).
To isolate and establish the structure of a product, biotransformation of δ-bromoγ-lactone 5 (100 mg) was carried out using P. frequentans AM 359. Spectral data of this product, obtained in 88% isolated yield, were fully consistent with those obtained for δ-hydroxy-γ-lactone 8 formed during the bromolactonization of ester 3. The postulated mechanism of the formation of lactone 8 catalyzed by fungi suggests a tandem dehalogenation-translactonization process involving two simultaneous nucleophilic substitutions: bromine atom at C-5 is substituted by a carboxylic anion released as a result of nucleophilic attack of water at C-6. According to the mechanism of nucleophilic substitution S N 2, approaching the water molecule from the side opposite to the broken C-O bond in the lactone ring results in the formation of only one isomer of trans δ-hydroxy-γ-lactone (8) (Scheme 4).  To isolate and establish the structure of a product, biotransformation of δ-bromo-γlactone 5 (100 mg) was carried out using P. frequentans AM 359. Spectral data of this product, obtained in 88% isolated yield, were fully consistent with those obtained for δ-hydroxy-γ-lactone 8 formed during the bromolactonization of ester 3. The postulated mechanism of the formation of lactone 8 catalyzed by fungi suggests a tandem dehalogenationtranslactonization process involving two simultaneous nucleophilic substitutions: bromine atom at C-5 is substituted by a carboxylic anion released as a result of nucleophilic attack of water at C-6. According to the mechanism of nucleophilic substitution SN2, approaching the water molecule from the side opposite to the broken C-O bond in the lactone ring results in the formation of only one isomer of trans δ-hydroxy-γ-lactone (8) (Scheme 4). Analysis of the composition of the products mixtures by GC showed the presence of two isomeric hydroxylactones 7 and 8. The percentage composition of the reaction mixtures indicated that initially only hydroxylactone 8 was formed and only after 3 or 7 days of the process the formation of hydroxylactone 7 was observed, the amount of which gradually increased at the expense of lactone 8. Such a course of biotransformation can be explained by the fact that the studied strains possess a dehydrogenase catalyzing a reversible oxidation of hydroxylactone 8 to the corresponding δ-keto-γ-lactone and fast reduction of the carbonyl group leading to the formation of diastereoisomeric hydroxylactone 7 (Scheme 5). A similar reversible oxidation/reduction activity has been described for the fungi-mediated biotransformations of alkylsubstituted cyclohexanones [28] or chalcones [29].  Analysis of the composition of the products mixtures by GC showed the presence of two isomeric hydroxylactones 7 and 8. The percentage composition of the reaction mixtures indicated that initially only hydroxylactone 8 was formed and only after 3 or 7 days of the process the formation of hydroxylactone 7 was observed, the amount of which gradually increased at the expense of lactone 8. Such a course of biotransformation can be explained by the fact that the studied strains possess a dehydrogenase catalyzing a reversible oxidation of hydroxylactone 8 to the corresponding δ-keto-γ-lactone and fast reduction of the carbonyl group leading to the formation of diastereoisomeric hydroxylactone 7 (Scheme 5). A similar reversible oxidation/reduction activity has been described for the fungi-mediated biotransformations of alkylsubstituted cyclohexanones [28] or chalcones [29]. Scheme 5. Transformation of γ-bromo-δ-lactone 5 to δ-hydroxy-γ-lactone 8 and subsequent formation of δ-hydroxy-γ-lactone 7 via reversible oxidation/reduction in the culture of A. cylindrospora AM 336 and D. igniaria KCH 6670.
Hydrolytic dehalogenation of halolactones catalyzed by A. cylindrospora AM 336 according to the S N 2 mechanism was observed for bicyclic δ-iodo-γ-lactones with unsubstituted, 4-methyl-, 4,4-dimethyl-, and 5,5-dimethylsubstituted cyclohexane ring as well as for β-phenyl-δ-iodo-γ-lactone. The process was highly stereospecific and the hydroxy group was introduced into the molecule from the opposite site to the leaving halogen atom which was clearly proven by 1 H NMR and X-ray data [32].
The process of dehalogenation via S N 2 mechanism with simultaneous translactonization leading to the formation of δ-hydroxy-γ-lactone, analogous to that observed in this work, was also reported during incubation of 3-methylcrotonaldehyde-derived γ-bromo-δ-lactone and γ-chloro-δ-lactone with Fusarium culmorum AM 3/1 and Rhodotorula rubra AM4, respectively [33]. Another case of tandem translactonization-dehalogenation reaction sequence was observed during biotransformation of δ-iodo-γ-lactone with 4,4-dimethylsubstituted cyclohexane ring by A. cylindrospora AM 336. In this case, the hydroxy group introduced by the fungi in the first step of transformation was involved in the intramolecular translactonization followed by immediate nucleophilic substitution by the iodine atom which resulted in the formation of γ,δ-epoxy-γ-lactone [32].

Antimicrobial Activity of Chalcone 1 and Lactones 5-8
Antibacterial and antifungal activity of chalcone 1 and lactones 5-8 were assessed based on duration of lag-phase (Tables 4 and 5) and changes in the optical density (∆OD) of microorganisms growing in the presence of tested compounds (Figures 5 and 6). The ability of a compound to limit the growth of a microorganism is manifested by a prolonged lag phase and/or a significantly lower biomass growth, expressed as ∆OD. Values of these two parameters were compared with those measured for microbial cultures growing without the studied compounds (control cultures). With complete inhibition of the microorganism's growth, there is no logarithmic growth phase, nor can the duration of the lag phase be determined; these cases are marked in Tables 4 and 5 as "not determinable". The tests were carried out for three strains of pathogenic bacteria (Escherichia coli, Bacillus subtilis, Staphylococcus aureus), three strains of filamentous fungi (Fusarium graminearum, Aspergillus niger, Alternaria sp.), and one strain of yeast (Candida albicans). Activity of the compounds was tested at the concentration of 0.1% in dimethyl sulfoxide (10 µL, w/v). Table 4. Effect of chalcone 1 and chalcone-derived lactones 5-8 on the duration of lag-phase of selected bacterial strains.

Compound
Escherichia coli PCM 2560  The results presented in Table 4 and Figure 5 indicate that for all bacterial strains the inhibitory effect of the studied compounds was observed. The highest activity was observed for bromolactone 6 and hydroxylactone 8 against S. aureus whose growth was completely inhibited. Relatively high activity towards this strain was also observed for bromolactone 5 and chalcone 1. In the case of B. subtilis, noticeable inhibitory properties were found for chalcone 1, bromolactone 6, and hydroxylactone 8 and the least active were bromolactone 5 and hydroxylactone 7. The most resistant bacterial strain was E. coli since in this case, only chalcone 1 significantly extended the lag-phase and mostly decreased biomass growth. Tested lactones influenced only on the latter parameter, and the lowest ΔOD was detected for bromolactone 6.  The results presented in Table 4 and Figure 5 indicate that for all bacterial strains the inhibitory effect of the studied compounds was observed. The highest activity was observed for bromolactone 6 and hydroxylactone 8 against S. aureus whose growth was completely inhibited. Relatively high activity towards this strain was also observed for bromolactone 5 and chalcone 1. In the case of B. subtilis, noticeable inhibitory properties were found for chalcone 1, bromolactone 6, and hydroxylactone 8 and the least active were bromolactone 5 and hydroxylactone 7. The most resistant bacterial strain was E. coli since in this case, only chalcone 1 significantly extended the lag-phase and mostly decreased biomass growth. Tested lactones influenced only on the latter parameter, and the lowest ∆OD was detected for bromolactone 6.  The compound with highest antifungal activity (Table 5, Figure 6) turned out to be hydroxylactone 8 which completely inhibited the growth of F. graminearum, A. niger, and Alternaria sp. and was highly active towards yeast C. albicans. Its isomer hydroxylactone 7 also totally inhibited the growth of Alternaria sp. and significantly limited the growth of A. niger but no activity of this compound was found against F. graminearum and C. albicans. Complete inhibition of microbial growth of A. niger, Alternaria sp., and C. albicans as well as significant limitation of the growth of F. graminearum was also observed for chalcone 1. For bromolactone 6, noticeably high inhibitory activity was found towards F. graminearum and Alternaria sp. and lower against A. niger, whereas diastereoisomeric bromolactone 5 was particularly active against F. graminearum and little active against A. niger. Interestingly, a few cases growth-promoting properties of tested compounds were found as ΔOD values were higher than those determined in control cultures. This phenomenon was particularly observed for bromolactones 6 and 5 incubated with C. albicans. The latter also promoted the growth of Alternaria sp. and hydroxylactone 7 slightly stimulated the growth of F. graminearum.

Bacillus subtilis B5
Considering the effect of the introduction of lactone ring into the chalcone scaffold on the antimicrobial activity one can see that a markedly higher activity in comparison with chalcone 1 was observed in the tests against S. aureus (Table 4, Figure 5) and F. graminearum (Table 5, Figure 6) for bromolactone 6 and hydroxylactone 8. In the tests against the latter strain, a clear increase in the activity was also observed for bromolactone 5. Compared to chalcone 1, lower ΔOD values were observed for bromolactone 6 against E. coli and hydroxylactone 8 against B. subtlilis ( Figure 5) although the durations of lag-phase determined in these tests were much shorter than those obtained for chalcone 1 (Table 4). In the tests against Alternaria sp., the activities of hydroxylactones 7 and 8 were as high as The compound with highest antifungal activity (Table 5, Figure 6) turned out to be hydroxylactone 8 which completely inhibited the growth of F. graminearum, A. niger, and Alternaria sp. and was highly active towards yeast C. albicans. Its isomer hydroxylactone 7 also totally inhibited the growth of Alternaria sp. and significantly limited the growth of A. niger but no activity of this compound was found against F. graminearum and C. albicans. Complete inhibition of microbial growth of A. niger, Alternaria sp., and C. albicans as well as significant limitation of the growth of F. graminearum was also observed for chalcone 1. For bromolactone 6, noticeably high inhibitory activity was found towards F. graminearum and Alternaria sp. and lower against A. niger, whereas diastereoisomeric bromolactone 5 was particularly active against F. graminearum and little active against A. niger. Interestingly, a few cases growth-promoting properties of tested compounds were found as ∆OD values were higher than those determined in control cultures. This phenomenon was particularly observed for bromolactones 6 and 5 incubated with C. albicans. The latter also promoted the growth of Alternaria sp. and hydroxylactone 7 slightly stimulated the growth of F. graminearum.
Considering the effect of the introduction of lactone ring into the chalcone scaffold on the antimicrobial activity one can see that a markedly higher activity in comparison with chalcone 1 was observed in the tests against S. aureus (Table 4, Figure 5) and F. graminearum (Table 5, Figure 6) for bromolactone 6 and hydroxylactone 8. In the tests against the latter strain, a clear increase in the activity was also observed for bromolactone 5. Compared to chalcone 1, lower ∆OD values were observed for bromolactone 6 against E. coli and hydroxylactone 8 against B. subtlilis ( Figure 5) although the durations of lag-phase determined in these tests were much shorter than those obtained for chalcone 1 (Table 4). In the tests against Alternaria sp., the activities of hydroxylactones 7 and 8 were as high as the ones found for chalcone 1; the same situation was observed comparing the activity of chalcone 1 and hydroxylactone 8 against A. niger. (Table 5, Figure 6). In other cases, the antifungal activities of lactones were lower than activity of chalcone 1.

Microorganisms Used for Biotransformation of Bromolactone 5
Strains of bacteria, yeast, and filamentous fungi used in biotransformations are listed in Tables 2 and 3 Strains with abbreviation AM came from Wroclaw Medical University. Microorganisms were stored on Sabouraud agar slants, pH 5.7, containing 1% peptone, 4% glucose, and 8% agar at 4 • C.
The progress of chemical reactions and biotransformations was checked by Gas Chromatography on an Agilent Technologies 6890N instrument with a flame ionization detector (FID) and hydrogen as a carrier gas. Compounds were analyzed on capillary column DB-5HT (30 m × 0.32 mm × 0.10 µm) using temperature program as follows: injector 200 • C, detector 280 • C, column temperature: 140 • C, 140-360 • C (rate 30 • C/min), and 360 • C (hold 1 min).
Single-crystal X-ray diffraction data were collected at 293 K (5), 100 K (6), and 150 K (7) on Xcalibur (Sapphire2 CCD detector for 5 and 7 or Onyx CCD detector for 6 κ-geometry diffractometers using Mo Kα (5 and 7) or Cu Kα radiation (6). Data reduction and analysis were carried out with the CrysAlis Pro programs (CrysAlis PRO. Versions: 1.171.36.28 or 1.171.33.66, currently Rigaku Oxford Diffraction, 2020). The structures were solved by direct methods and refined with the full-matrix least-squares technique using the SHELXS [34] and SHELXL [35] programs. Non-hydrogen atoms were refined with anisotropic displacement parameters. All H atoms were placed at calculated positions. Before the last cycle of refinement, all H atoms were fixed and were allowed to ride on their parent atoms.
Crystal data for 5, 6, and 7 reported in this paper have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication numbers 2,254,514, 2,254,515 and 2,254,516, respectively. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB12 1EZ, UK (fax +44-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
Uncorrected melting points were determined on Boetius apparatus.

Synthesis of Lactones 5-8 3.4.1. Reduction of Chalcone (1)
A solution of chalcone (1) (10.2 g, 49 mmol) in 100 mL of methanol was placed in an ice bath on a magnetic stirrer. Then, an aqueous solution (10 mL) of sodium borohydride (2.35 g, 0.049 mol) was added dropwise and the reaction mixture was stirred by 6 h and after that it was transferred into a separatory funnel, diluted with 25 mL of hot water and the product was extracted with methylene chloride (3 × 40 mL). The combined organic layers were washed with saturated sodium chloride solution until neutral and dried with anhydrous MgSO 4 . The organic solvent was evaporated on a vacuum evaporator to obtain pure alcohol 2. (

Claisen Rearrangement of Alcohol 2
A mixture of alcohol 2 (8.63 g; 41 mmol), triethyl orthoacetate (60 mL; 0.33 mol), and propionic acid (two drops) was heated at 138 • C in a two-necked round-bottom flask fitted with a distillation cap. When the reaction was finished (13 h, TLC, GC), the excess of triethyl orthoacetate was distilled off and the crude product was purified by column chromatography (hexane:acetone, 40:1) to afford known [36]

Bromolactonization of Acid 4
A solution of acid 4 (0.104 g, 40 mmol), NBS (0.082 g, 0.46 mmol), and a drop of acetic acid in THF (20 mL) was stirred at room temperature. When the substrate was consumed (36 h, TLC, GC), the reaction mixture was extracted with diethyl ether (3 × 40 mL), followed by washing with saturated NaHCO 3 solution and brine. The organic extracts were dried with anhydrous MgSO 4 and the solvent was evaporated on a vacuum evaporator. The crude product was purified by column chromatography (hexane:acetone, 10:1) to afford known [23] bromolactone 5.
5-t-Bromo-4(r),6(c)-diphenyltetrahydropyran-2-one (5): yield 37% (0.051 g), white crystals, mp 176-178 • C, 1  The strains were cultivated on rotary shakers (144 rpm) at 25 • C in 300 mL Erlenmayer flasks containing 50 mL of medium (3% glucose, 1% peptone, pH 6.2). After 5 days, 10 mg of bromolactone 5 dissolved in 1 mL of acetone was added to each flask and the incubation of shaken cultures with substrate was continued for 14 days. Biotransformation products were extracted with ethyl acetate after 1, 3, 7, 10, and 14 days. The extracts were dried with anhydrous magnesium sulphate, concentrated on a rotary evaporator, dissolved in methanol, filtered through a syringe filter (13 mm × 0.45 µm), and analyzed by TLC and GC HPLC. The stability of the substrate was also checked under biotransformation conditions, and the pH of the substrates was tested during the processes. In order to identify the metabolites secreted by individual microbial strains, microorganisms were also cultivated without substrate addition. The results of screening procedure are presented in Tables 2 and 3. 3.5.2. Biotransformation of Bromolactone 5 by P. frequentans AM 359 Bromolactone 5 (100 mg dissolved in 10 mL of acetone) was added to the 5-day cultures of P. frequentans AM 359 prepared as described in the screening procedure. The culture was shaken in 2L flask with 400 mL of medium. The progress of biotransformation was monitored by GC. After 7 days, the product was extracted with ethyl acetate. The organic fractions were dried with anhydrous MgSO 4 and the solvent was evaporated on a vacuum evaporator. Column chromatography (hexane:ethyl acetate; 7:1) afforded 71 mg (yield 88%) of pure hydroxylactone 8 with physical and spectral data consistent with those given in Section 3.4.5.

Antimicrobial Activity Assay
Antimicrobial tests were carried out using the strains from the collection of the Department of Biotechnology and Food Microbiology, Wroclaw University of Environmental and Life Sciences: bacteria Escherichia coli PCM 2560, Staphylococcus aureus D1, and Bacillus subtilis B5, filamentous fungi Fusarium graminearum 109, Alternaria sp., and Aspergillus niger XP, and yeast Candida albicans KL-1.
The tests were carried out on the automated Bioscreen C system (Automated Growth Curve Analysis System, Lab Systems, Finland) according to the procedure described in our previous paper [21]. Tested compounds were applied as 0.1% solutions in 10 µL of dimethyl sulfoxide (DMSO) (w/v). The results presented in Figures 5 and 6 were analyzed using spreadsheet software (Excel 97). Statistics on a completely randomized design were determined using the one-way analysis of variance (ANOVA) procedure at a level of significance set at p < 0.050. Dunnett's test was used to compare the average ∆OD of microorganism growth in the presence of tested compounds relative to the control.

Conclusions
In this work, synthetic and biocatalytic approach to chalcone-derived lactones was presented. Bromolactonization of ester 3 with NBS carried out in the mixture of THF and water, in addition to the known γ-bromo-δ-lactone 5, also gives access to three unexpected products (Scheme 2). The first one is diastereoisomeric γ-bromo-δ-lactone 6 in which a bromine at C-5 and a phenyl ring at C-6 occupy the pseudoaxial positions at the sixmembered lactone ring. Diastereoisomeric trans δ-hydroxy-γ-lactones 7 and 8 differing in the relative configuration of hydroxy group at C-6 were also isolated. Formation of these two hydroxylactones during bromolactonization of ester 3 is the result of S N 1 type of nucleophilic substitution involving a facilitated dissociation of the bromine at the benzyl position of primarily formed trans δ-bromo-γ-lactone and fast reaction of the benzylic carbocation with the water present in the reaction medium (Scheme 3).
Most of the fungal strains used for the biotransformation of γ-bromo-δ-lactone 5 have the ability to transform the substrate to only one product, trans δ-hydroxy-γ-lactone 8, in the tandem dehalogenation-translactonization process involving two simultaneous S N 2 nucleophillic substitutions (Scheme 4). Only A. cylindrospora AM 336 and D. igniaria KCH 6670 produced both isomers of δ-hydroxy-γ-lactones 7 and 8 by reversible oxidation/reduction activity (Scheme 5).
Selective formation of trans δ-hydroxy-γ-lactone 8 by P. frequentans-mediated biotransformation in relatively high yield (88%) is of special interest because this compound exhibited the most potent inhibitory activity towards tested microorganisms. It was particularly active against fungal strains F. graminearum, A. niger, and Alternaria sp. The most positive effect of the lactone moiety introduced into the scaffold of chalcone 1 on the antibacterial activity was demonstrated towards S. aureus by δ-hydroxy-γ-lactone 8 and γ-bromo-δ-lactone 6; a similar effect on the antifungal activity for these two lactones and γ-bromo-δ-lactone 5 was shown towards F. graminearum.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.